**8. Presence of DNA damage by comet assay as genotoxicity biomarker**

We treated fibroblast cells with three different doses of a mixture of benzo(a)pyrene and beta-naphthoflavone for 4 h. The viability of fibroblast cells, assessed by the trypan blue test, was very high in control (>95%) and slightly decreased following the different treatments. We analysed DNA damage by the Comet assay. The principle of the Comet assay is that smaller DNA molecules migrate faster in an electric field than larger molecules. The treated cells are encapsulated in gel and lysed by alkali, which also denatures the DNA. Subsequent electrophoresis causes migration of the DNA. While the undamaged DNA appears as a "head", fragmented DNA move faster, giving the characteristic appearance of a comet tail. Figure 9 shows four normal cells at increasing degree of damaged DNA.

The cells were processed at different conditions of electrophoresis, to evaluate different types of strand breaks. Varying the pH during lysis and electrophoresis effects the type of strand breaks expressed. When cells are lysed and subjected to electrophoresis under neutral conditions (pH 8) only double strand breaks were detected. Under pH 12.1 conditions the double and single strand breaks were detected while under pH > 13 conditions the double strand breaks, single strand breaks and alkali labile lesions were detected [44].

The results of Comet assay evaluated in cells processed at pH 12.1 are shown in Figure 10. An increase of DNA migration was observed in fibroblasts exposed for 4 h at doses C and B, while a slight decrease was observed at dose A in comparison to the control. The acetone, used as carrier for benzo(a)pyrene and beta-naphthoflavone exposure, showed DNA fragmentation values very similar to the control.

"Test Tube Cetaceans": From the Evaluation of Susceptibility to the Study of Genotoxic Effects of Different Environmental Contaminants Using Cetacean Fibroblast Cell Cultures 69

68 New Approaches to the Study of Marine Mammals

solvent control.

Only two species, the long-beaked common dolphin (MDC12) and the sperm whale (PMAS1) were treated with PAHs. However, even with this treatment, there is a significant dose/response type increase in the level of MICA in the sperm whale (PMAS1) (Figure 8C).

Table 11 shows the results of the mean levels of immunofluorescence of MICA, revealed in cultured fibroblasts of different species treated with the BPA, expressed as index numbers.

**RT23 (striped dolphin)** 100 84 99 85 D.C. **RT25 (fin whale)** 100 62 68 153 D.C.

**Table 11.** Mean values of immunofluorescence of MICA revealed in cultured fibroblasts of different species treated with BPA. The immunofluorescence is expressed in index numbers respect to solvent

The results showed that the higher dose caused the death of all cells, thus focusing all its toxicity. Only fin whale (RT25) fibroblasts had an inductive phenomenon with respect to

**8. Presence of DNA damage by comet assay as genotoxicity biomarker** 

We treated fibroblast cells with three different doses of a mixture of benzo(a)pyrene and beta-naphthoflavone for 4 h. The viability of fibroblast cells, assessed by the trypan blue test, was very high in control (>95%) and slightly decreased following the different treatments. We analysed DNA damage by the Comet assay. The principle of the Comet assay is that smaller DNA molecules migrate faster in an electric field than larger molecules. The treated cells are encapsulated in gel and lysed by alkali, which also denatures the DNA. Subsequent electrophoresis causes migration of the DNA. While the undamaged DNA appears as a "head", fragmented DNA move faster, giving the characteristic appearance of a comet tail.

The cells were processed at different conditions of electrophoresis, to evaluate different types of strand breaks. Varying the pH during lysis and electrophoresis effects the type of strand breaks expressed. When cells are lysed and subjected to electrophoresis under neutral conditions (pH 8) only double strand breaks were detected. Under pH 12.1 conditions the double and single strand breaks were detected while under pH > 13 conditions the double

The results of Comet assay evaluated in cells processed at pH 12.1 are shown in Figure 10. An increase of DNA migration was observed in fibroblasts exposed for 4 h at doses C and B, while a slight decrease was observed at dose A in comparison to the control. The acetone, used as carrier for benzo(a)pyrene and beta-naphthoflavone exposure, showed DNA

*0.1% 0.1 µg/ml 1 µg/ml 10 µg/ml 100 µg/ml* 

Long-beaked common dolphin (MDC12) showed no induction response.

**7.5. MICA in different species after treatment with BPA** 

control. Different colour of box is related to different increase of this protein.

Figure 9 shows four normal cells at increasing degree of damaged DNA.

strand breaks, single strand breaks and alkali labile lesions were detected [44].

fragmentation values very similar to the control.

*MICA Ethanol* 

**Figure 9.** A photomicrograph of fibroblast cells of striped dolphin processed for the DNA comet assay. Four normal cells at increasing degree of damaged DNA: normal DNA, low-level DNA damage, DNA with a long tail, DNA almost completely fragmented.

The highest DNA fragmentation was observed at dose C and decreasing DNA tail values were observed from dose C to dose A. The results of the Comet assay evaluated in striped dolphin fibroblast cells processed at three different pHs showed a similar trend (Table 12), although the trend was more evident for the pH 12.1. Our results, although preliminary, suggest that alkaline Comet assay (pH 12.1) is the optimal version capable of detecting the DNA damage in fibroblast cells for future analysis.


"Test Tube Cetaceans": From the Evaluation of Susceptibility to the Study of

Genotoxic Effects of Different Environmental Contaminants Using Cetacean Fibroblast Cell Cultures 71

in these marine mammals. The data reported in this chapter confirm that the use of Test Tube Cetaceans is a good non destructive surrogate of "in vivo" cetacean test (killing) to

Regarding the toxicological susceptibility to some xenobiotic compounds and to PAHs, the main results showed that the basal level of CYP1A1 and CYP2B of different cetacean species is very dissimilar and this seems to be especially species-specific rather than related to the geographic range, diet, toxicological status, etc. in which the specimens were found. All pollutants, at different level depending on the species and of the dose of treatment, showed an inductive capacity of these cytochromes. At times the response was dose dependent,

The qualitative and quantitative MICA protein expression as toxicological stress marker of the immune system showed that the three species sampled in the Sea of Cortez (Bryde's whale, long-beaked common dolphin and killer whale) showed higher basal level of MICA in respect to all Mediterranean specimens, regardless of the species and the fact that they were sampled free-ranging or found stranded alive and then died. This sharp distinction between the activity of MICA found in the Sea of Cortez and Mediterranean Sea specimens is probably the most important result to be highlighted: it seems that the environment in which specimens live and, therefore, the anthropogenic stress to which they are subjected, are determinant in the response of this protein of the immune system. In the light of this result we can hazard the conclusion that the lower the anthropic stress of the specimens, the

The Comet assay proved to be a very useful tool for assessing the potential genotoxicity of PAHs in cetacean fibroblast cell cultures. Future investigations will be conducted to investigate the genotoxic effects of different classes of contaminants in striped dolphin and other cetacean species. This technique led to the evaluation of possible DNA damage in species never studied before in this field, in order to investigate the different susceptibility

In conclusion, "Test Tube Cetaceans" can be proposed to the scientific community as the "in vitro" method used to replace the "scientific whaling" to study the toxicological threats of different species of cetaceans, primarily of endangered species such as fin whale and Mediterranean common dolphin, since the research priorities is the conservation for the

, Silvia Maltese, Daniele Coppola, Ilaria Caliani, Laura Carletti,

Matteo Giannetti, Tommaso Campani, Matteo Baini, Cristina Panti, Silvia Casini

*Department of Environmental Sciences, University of Siena, Siena, Italy* 

other was bell-shaped response and other was a discontinuous induction response.

evaluate the different hazards of cetaceans to pollution.

higher is the basal level of MICA.

maintenance of cetacean biodiversity.

**Author details** 

and M. Cristina Fossi

Corresponding Author

Letizia Marsili\*

 \*

to various contaminants, using fibroblast cell cultures.

**Table 12.** DNA migration evaluated in fibroblast cells of striped dolphin at three different pH conditions.

**Figure 10.** Effects of different doses of the mixture benzo(a)pyrene and beta-naphthoflavone at three doses (C = (0.5μM BaP + 10μM BnF), B = (2.5μM BaP + 50μM BnF) and A = (12.5μM BaP + 250μM BnF)) exposure on the DNA integrity of striped dolphin fibroblast cells after 4 h exposure.

The induction of DNA fragmentation was higher at the lowest dose (Dose C), while decreased at higher doses (Dose B and A), a result in contrast to other studies on cell mammal cultures [45, 46]. These investigations have demonstrated that an increase in the percentage of DNA in the tail region of the comets occurred in a concentration-dependent manner after exposure to different classes of genotoxic compounds, such as PAHs, methyl methanesulfonate (MMS) and H2O2. Our earlier Comet assay data on dolphin fibroblast cells exposed to benzo(a)pyrene are in agreement with the bibliography data. Thus, the decrease observed in our present data could be probably due to the action of the betanaphthoflavone. This hypothesis is consistent with Gravato *et al.* [47] who demonstrated a decrease of DNA damage after exposure of specimens of *Anguilla anguilla* to betanaphthoflavone. However, further studies are needed to confirm the genotoxic potential of mixture of PAHs for cetacean fibroblasts and investigate the potential genotoxicity of other classes of contaminants.
